Identification and mutation of a gene required for glycerate kinase activity from a facultative methylotroph, Methylobacterium extorquens AM1

Chistoserdova, L.; Lidstrom, Mary E.

doi:10.1128/jb.179.15.4946-4948.1997

Cited by 23 publications

(29 citation statements)

References 22 publications

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“…This type of enzyme is widely present in all kingdoms of living organisms and plays an important function in cell metabolism. Correct functioning of such enzymes is necessary for the C 2 respiratory cycle in Arabidopsis thaliana (2) and has a role in serine metabolism in Methylobacterium extorquens (8). In humans, a deficiency of glycerate kinase leads to D-glyceric aciduria, which is characterized by massive amounts of D-glyceric acid in urine (3).…”

Section: Discussionmentioning

confidence: 99%

Influence of the σ^BStress Factor andyxaB, the Gene for a Putative Exopolysaccharide Synthase under σ^BControl, on Biofilm Formation

et al. 2008

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Bacillus subtilis forms structured communities of biofilms encased in an exopolysaccharide matrix on solid surfaces and at the air-liquid interface. It is postulated that nonoptimal growth conditions induce this multicellular behavior. We showed that under laboratory conditions a strain deleted for sigB was unable to form a floating pellicle on the surface of a liquid medium. However, overexpression of yxaB, encoding a putative exopolysaccharide synthase, from a p Spac promoter in a sigB-deleted strain resulted in partial recovery of the wild-type phenotype, indicating the participation of the YxaB protein in this multicellular process. We present data concerning the regulation of transcription of genes yxaB and yxaA, encoding a putative glycerate kinase. Both genes are cotranscribed as a single transcription unit from a A -dependent promoter during vegetative growth of a liquid bacterial culture. The promoter driving transcription of the yxaAB operon is regulated by AbrB. In addition, the second gene in the operon, yxaB, possesses its own promoter, which is recognized by RNA polymerase containing the B subunit. This transcription start site is used under general stress conditions, resulting in increased expression.Since bacteria in their natural setting face widely fluctuating conditions, including changes in pH, temperature, or limitation of nutrients, biofilms appear to be an excellent adaptive strategy to protect the inhabitants from diverse stress factors (11). Indeed, it is commonly known that the biofilm mode of growth is the major bacterial life-style in nature (9). In order to adapt to a wide diversity of stresses, Bacillus subtilis uses an alternative sigma factor, B , to express a large set of genes in the B regulon. The B factor is estimated to control at least 5% of the coding capacity of the genome (36).In B. subtilis, 17 sigma factors have been described, showing a wide range of recognition sequences. The major vegetative sigma factor is required for expression of so-called housekeeping genes, whereas exposure of bacteria to a wide variety of growth-limiting conditions requires the alternative sigma factor, B (17, 19, 36). On the other hand, changes in gene expression that occur during the transition from a planktonic state to a biofilm mode of growth depend on several sigma factors associated mainly with sporulation (4, 15, 38, 39). Despite the fact that a large number of sporulation genes are induced in the wild-type biofilm, the forespore sigma factor F and its counterpart E , which is activated in the mother cell, seem not to play any role in biofilm formation (4,15,38). Surprisingly, the absence of H , which is involved in the early stages of sporulation, nevertheless does affect this process. The most characteristic defect associated with the lack of H is the absence of fruiting-body structures typical of mature biofilms of natural isolates of B. subtilis (4). Recent data showed that a strain with inactivated sigma factors X , M , and W displayed a complete loss of the pellicle formation (26...

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Section: Discussionmentioning

confidence: 99%

Influence of the σ^BStress Factor andyxaB, the Gene for a Putative Exopolysaccharide Synthase under σ^BControl, on Biofilm Formation

et al. 2008

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“…(iv) 13 C labeling. The incubation of cells with uniformly labeled glyoxylate (1,2-13 C; 99%; Cambridge Isotope Laboratories, Andover, MA) was carried out at 28°C in a 25-ml glass tube containing 9.5 l of 0.532 M labeled glyoxylate.…”

Section: Methodsmentioning

confidence: 99%

Alternative Route for Glyoxylate Consumption during Growth on Two-Carbon Compounds by Methylobacterium extorquens AM1

Okubo¹,

Yang²,

Chistoserdova³

et al. 2010

J Bacteriol

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Methylobacterium extorquens AM1 is a facultative methylotroph capable of growth on both single-carbon and multicarbon compounds. Mutants defective in a pathway involved in converting acetyl-coenzyme A (CoA) to glyoxylate (the ethylmalonyl-CoA pathway) are unable to grow on both C 1 and C 2 compounds, showing that both modes of growth have this pathway in common. However, growth on C 2 compounds via the ethylmalonylCoA pathway should require glyoxylate consumption via malate synthase, but a mutant lacking malyl-CoA/ ␤-methylmalyl-CoA lyase activity (MclA1) that is assumed to be responsible for malate synthase activity still grows on C 2 compounds. Since glyoxylate is toxic to this bacterium, it seemed likely that a system is in place to keep it from accumulating. In this study, we have addressed this question and have shown by microarray analysis, mutant analysis, metabolite measurements, and 13 C-labeling experiments that M. extorquens AM1 contains an additional malyl-CoA/␤-methylmalyl-CoA lyase (MclA2) that appears to take part in glyoxylate metabolism during growth on C 2 compounds. In addition, an alternative pathway appears to be responsible for consuming part of the glyoxylate, converting it to glycine, methylene-H 4 F, and serine. Mutants lacking either pathway have a partial defect for growth on ethylamine, while mutants lacking both pathways are unable to grow appreciably on ethylamine. Our results suggest that the malate synthase reaction is a bottleneck for growth on C 2 compounds by this bacterium, which is partially alleviated by this alternative route for glyoxylate consumption. This strategy of multiple enzymes/pathways for the consumption of a toxic intermediate reflects the metabolic versatility of this facultative methylotroph and is a model for other metabolic networks involving high flux through toxic intermediates.Methylobacterium extorquens AM1 grows on one-carbon (C 1 ) compounds using the serine cycle for assimilation (25). This metabolism requires the conversion of acetyl-coenzyme A (CoA) to glyoxylate, which occurs via a novel pathway in which acetyl-CoA is converted to methylsuccinyl-CoA via acetoacetyl-CoA, ß-hydroxybutyryl-CoA, and ethylmalonyl-CoA (30-33). Recently, the steps involved in the conversion of methylsuccinyl-CoA to glyoxylate have been elucidated, and the pathway has been termed the ethylmalonyl-CoA (EMC) pathway (1,19,20,40). Careful labeling measurements coupled to measurements of intermediates has confirmed that, during the growth of M. extorquens AM1 on methanol, methylsuccinyl-CoA is converted to glyoxylate and propionyl-CoA via mesaconyl-CoA and ß-methylmalyl-CoA (40).This finding has raised questions regarding how M. extorquens AM1 grows on two-carbon (C 2 ) compounds. The pathway involved in the conversion of acetyl-CoA to glyoxylate is known to operate during growth on both C 1 and C 2 compounds, as mutants in genes involved in this conversion are unable to grow on either C 1 or C 2 compounds, and in both cases they are rescued by glyoxylate (11,(15)(16)(17)44). ...

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“…Methylotrophy in M. extorquens AM1 is well-understood at the physiological and genetic levels and tools are available for genetic manipulations, making it an attractive system for the development of metabolic engineering techniques in methylotrophs. Eighty-six genes have been characterized, most of which are involved in the methanol assimilation and oxidation pathways unique to methylotrophic bacteria (Chistoserdova, 1996). In contrast, little is known about the growth of M. extorquens AM1 on C 3 and C 4 compounds, or of the metabolism of multicarbon compounds once they are produced from formaldehyde via the serine cycle.…”

Section: Introductionmentioning

confidence: 99%

“…One of the most widely studied of the methanolutilizing bacteria is the pink-pigmented facultative methylotroph Methylobacterium extorquens AM1. This organism is capable of growth on a variety of C 2 , C 3 and C 4 compounds as well as methanol and methylamine, assimilates methanol at the level of formaldehyde by the serine cycle and has served as the primary model system for the study of methylotrophic metabolism and methylotrophic enzymes (Chistoserdova, 1996;Lidstrom, 1992). Methylotrophy in M. extorquens AM1 is well-understood at the physiological and genetic levels and tools are available for genetic manipulations, making it an attractive system for the development of metabolic engineering techniques in methylotrophs.…”

Section: Introductionmentioning

confidence: 99%

Reconstruction of C3 and C4 metabolism in Methylobacterium extorquens AM1 using transposon mutagenesis

et al. 2003

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The growth of Methylobacterium extorquens AM1 on C 1 compounds has been well-studied, but little is known about how this methylotroph grows on multicarbon compounds. A Tn5 transposon mutagenesis procedure was performed to identify genes involved in the growth of M. extorquens AM1 on succinate and pyruvate. Of the 15 000 insertion colonies screened, 71 mutants were found that grew on methanol but either grew slowly or were unable to grow on one or both of the multicarbon substrates. For each of these mutants, the chromosomal region adjacent to the insertion site was sequenced, and 55 different genes were identified and assigned putative functions. These genes fell into a number of predicted categories, including central carbon metabolism, carbohydrate metabolism, regulation, transport and non-essential housekeeping functions. This study focused on genes predicted to encode enzymes of central heterotrophic metabolism: 2-oxoglutarate dehydrogenase, pyruvate dehydrogenase and NADH : ubiquinone oxidoreductase. In each case, the mutants showed normal growth on methanol and impaired growth on pyruvate and succinate, consistent with a role specific to heterotrophic metabolism. For the first two cases, no detectable activity of the corresponding enzyme was found in the mutant, verifying the predictions. The results of this study were used to reconstruct multicarbon metabolism of M. extorquens AM1 during growth on methanol, succinate and pyruvate.

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Identification and mutation of a gene required for glycerate kinase activity from a facultative methylotroph, Methylobacterium extorquens AM1

Cited by 23 publications

References 22 publications

Influence of the σ^BStress Factor andyxaB, the Gene for a Putative Exopolysaccharide Synthase under σ^BControl, on Biofilm Formation

Influence of the σ^BStress Factor andyxaB, the Gene for a Putative Exopolysaccharide Synthase under σ^BControl, on Biofilm Formation

Alternative Route for Glyoxylate Consumption during Growth on Two-Carbon Compounds by Methylobacterium extorquens AM1

Reconstruction of C3 and C4 metabolism in Methylobacterium extorquens AM1 using transposon mutagenesis

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Identification and mutation of a gene required for glycerate kinase activity from a facultative methylotroph, Methylobacterium extorquens AM1

Cited by 23 publications

References 22 publications

Influence of the σBStress Factor andyxaB, the Gene for a Putative Exopolysaccharide Synthase under σBControl, on Biofilm Formation

Influence of the σBStress Factor andyxaB, the Gene for a Putative Exopolysaccharide Synthase under σBControl, on Biofilm Formation

Alternative Route for Glyoxylate Consumption during Growth on Two-Carbon Compounds by Methylobacterium extorquens AM1

Reconstruction of C3 and C4 metabolism in Methylobacterium extorquens AM1 using transposon mutagenesis

Contact Info

Product

Resources

About

Influence of the σ^BStress Factor andyxaB, the Gene for a Putative Exopolysaccharide Synthase under σ^BControl, on Biofilm Formation

Influence of the σ^BStress Factor andyxaB, the Gene for a Putative Exopolysaccharide Synthase under σ^BControl, on Biofilm Formation